The focus of this proposal is to extend the recently developed genetic systems that permit gene transfer in mycobacterial species in order to define genes responsible for the virulence of N. tuberculosis. Transposon mutagenesis systems will be developed for N. tuberculosis and BCG in order to generate libraries of insertional mutations. Homologous recombination has not been possible in M. tuberculosis and so strains and systems will be developed that permit the specific genetic engineering of M. tuberculosis. Novel screening systems will also be developed to identify the genes of M. tuberculosis that are induced when the bacilli are grown in animal hosts. The nature of the mutations that attenuate the well- characterized BCG strains and the M. tuberculosis H37Ra strain will be investigated using both genetic transfer and genomic mismatch scanning strategies. This work may provide the basis for defining virulence and drug resistance determinants of M. tuberculosis as well as the means for engineering novel tuberculosis vaccines.